Super-enhancer lncs to cardiovascular development and disease Samir Ounzain, Thierry Pedrazzini PII: DOI: Reference:

S0167-4889(15)00410-3 doi: 10.1016/j.bbamcr.2015.11.026 BBAMCR 17737

To appear in:

BBA - Molecular Cell Research

Received date: Revised date: Accepted date:

22 September 2015 20 November 2015 23 November 2015

Please cite this article as: Samir Ounzain, Thierry Pedrazzini, Super-enhancer lncs to cardiovascular development and disease, BBA - Molecular Cell Research (2015), doi: 10.1016/j.bbamcr.2015.11.026

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Super-Enhancer Lncs to Cardiovascular Development and Disease

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Samir Ounzain1*, Thierry Pedrazzini1* 1

Experimental Cardiology Unit, Department of Medicine, University of Lausanne Medical

Corresponding author:

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School, Switzerland

Dr Thierry Pedrazzini Experimental Cardiology Unit

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Department of Medicine

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University of Lausanne Medical School

Phone:

+41 21 314 0765

Fax:

+41 21 314 9477

Email:

[email protected]

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CH-1011 Lausanne, Switzerland

*Correspondence can be addressed to:

[email protected]; [email protected]

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ACCEPTED MANUSCRIPT Abstract Cardiac development, function and pathological remodelling in response to stress depends on the dynamic control of tissue specific gene expression by distant acting

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transcriptional enhancers. Recently, super-enhancers (SEs), also known as stretch or large enhancer clusters, are emerging as sentinel regulators within the gene regulatory networks

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that underpin cellular functions. It is becoming increasingly evident that long noncoding RNAs (lncRNAs) associated with these sequences play fundamental roles for enhancer

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activity and the regulation of the gene programs hardwired by them. Here, we review this emerging landscape, focusing on the roles of SEs and their derived lncRNAs in

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cardiovascular development and disease. We propose that exploration of this genomic landscape could provide novel therapeutic targets and approaches for the amelioration of cardiovascular disease. Ultimately we envisage a future of ncRNA therapeutics targeting the

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SE landscape to alleviate cardiovascular disease.

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ACCEPTED MANUSCRIPT 1. Introduction Coordinated temporal, spatial and signal dependant control of gene expression is critical for cardiovascular development, homeostasis, pathological remodelling and cardiac

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regeneration [1,2]. Decades of investigations have shed led on the plethora of transcription factors responsible for driving cardiac specification, patterning, differentiation, maturation

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and stress responses within the remodelling heart. Furthermore, recent studies have illuminated our understanding of how global epigenetic and transcriptional changes promote

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lineage determination during embryonic cardiovascular development, cardiac maturation and maladaptive pathological remodelling [1-3]. Fundamentally at the molecular gene regulatory

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level, these processes are under the control of the integrated activity of the core cardiac transcription factors (TFs) including MESP1, HAND2, TBX5, MEF2C, GATA4 and NKX2.5. These TFs interact in a combinatorial manner at target -cis regulatory sequences (i.e.

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proximal promoters and distal enhancers) in a context dependant manner to elicit the appropriate gene expression patterns. The binding of these TFs is coupled with the dynamic

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remodelling of the underlying chromatin which reprograms the epigenomic landscape and nuclear architecture ultimately driving the global gene expression patterns responsible for cell fate, phenotype and behaviour.

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The dynamic regulation of the epigenome, nuclear architecture, transcriptome and proteome is highly integrated to coordinate the outputs of otherwise disparate molecular networks [1-3]. This, for example, allows the considerable changes cardiac cells undergo in response to stress. This includes the transcriptional re-activation of the fetal gene program, hypertrophic growth of cardiomyocytes without cell division and metabolic state changes leading to the maladaptive pathological remodelling that drives the development of heart failure. Despite our advancing knowledge, the epigenetic and transcriptional reprogramming of the remodelling and failing heart is a complex process and the full spectrum of molecular determinants of these processes are waiting to be deciphered. Within this context it has recently emerged that the noncoding portion of the genome (genomic ‘dark matter’) encodes a vast repertoire of regulatory -cis sequences (enhancers) and associated noncoding RNAs

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ACCEPTED MANUSCRIPT (ncRNAs) with crucial regulatory functions within the gene regulatory networks that dictate cardiovascular development and disease [4,5]. In particular, transcripts derived from enhancers and super-enhancers (SEs) are emerging as interesting regulatory molecules in

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need of further characterisation [5]. This new understanding of the enhancer landscape and associated ncRNAs opens a new exciting area of enhancer therapy to modulate the gene

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regulatory networks underpinning cardiovascular disease in a highly targeted and efficient therapeutic manner [5]. In particular we propose that cardiac SEs and their associated long

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noncoding RNAs (lncRNAs) could represent highly attractive therapeutic targets that will be

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introduced and discussed in this short review.

2. Genomic ‘Dark Matter’

Gene regulatory networks were historically interpreted in a protein centric manner.

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However, recent global genomic approaches have begun to illuminate our understanding of genome biology and surprisingly demonstrated that only 2% of our genomic sequence

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actually codes for protein. The remaining 98% was previously described as junk DNA but is now more appropriately called genomic ‘dark matter’ [6,7]. This ‘dark matter’ is packed with cis regulatory enhancer sequences which are dynamically transcribed generating thousands

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of lncRNAs with functionally and structurally diverse regulatory properties [8,9]. Over the past few years many studies have demonstrated that these lncRNAs are partly responsible for highly complex patterns of gene regulation, which underpins specialised biological functions during embryonic development and in to adulthood [4,10]. Specifically, lncRNAs have been shown to control most aspects of gene regulatory network activity including epigenetic targeting, transcriptional control, nuclear genome organisation and post transcriptional processing [11]. Importantly it has been shown that lncRNA numbers scale proportionally, unlike coding genes, with developmental and cellular complexity within metazoans, supporting highly specialised important functional roles for these molecules [11]. Despite this, the roles of enhancers and their associated lncRNAs within the cardiovascular system remain to be fully defined [5]. Incorporating these functional sequences and

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ACCEPTED MANUSCRIPT transcripts within the logic governing cardiac gene regulatory networks will without doubt provide unprecedented insights and opportunities for developing novel therapeutic strategies for the amelioration of cardiovascular disease. This short review aims to highlight new

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concepts regarding the roles of genomic ‘dark matter’ in cardiovascular development and

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disease with a particular emphasis on the roles of SEs and their associated lncRNAs.

3. Cardiovascular Genetic Switches - Enhancers

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The biological processes governing cardiovascular development and homeostasis require cells to respond to developmental and environmental cues by executing specific

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transcriptional programmes from a single set of genetic material [12]. Distally located -cis regulatory sequences known as enhancers, are the primary information processing units within the genome that enable correct temporal and spatial execution of these programs.

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Enhancers were first described in the 1980s as -cis regulatory elements distal to genes, which, in contrast to promoters, modulated transcription in an orientation independent

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manner [13]. Within mammalian genomes, enhancers are located within the linear sequence, but appropriate communication with and expression of their genes requires the formation of 3D loops between enhancers and their target promoters [13]. Enhancers

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themselves are composed of clusters of transcription factor binding sites (TFBS) which when bound by lineage determining TFs (LDTFs) regulate gene expression in a context dependant manner. Bound cardiac TFs and recruited chromatin regulators coordinate the activation and repression of complex transcriptional gene programs that underpin the cardiovascular gene regulatory network [2,5]. A number of recently published epigenomic screens suggest that hundreds of thousands of putative enhancers exist within the human genome, vastly outnumbering protein coding genes [6]. Furthermore, typically each protein coding gene is potentially regulated by tens of enhancers creating highly complex and integrated -cis regulatory networks. Not surprisingly, these observations have led many to suggest that the complexity of the dynamically regulated enhancer landscape is responsible for the integrated

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ACCEPTED MANUSCRIPT gene expression patterns required for specialised developmental programs and adaption to pathological signals. Cardiovascular development in particular is associated with the dynamic regulation of

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the enhancer landscape where complex transcriptional patterns of gene expression are required for cardiac morphogenesis [2,5]. Not surprisingly, disruption of these cardiac gene

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regulatory networks at the level of the enhancer sequences, the bound cardiac TFs and the associated chromatin remodelling complexes underpins congenital heart defects and

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susceptibility to acquired adult heart pathologies [1,2]. To asses the enhancer landscape during development, a number of studies have utilised epigenomic screens to identify and

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characterise developmental cardiac enhancers. An early study identified approximately 3000 developmental cardiac enhancers in the murine whole heart at embryonic day 11.5 using ChIP-sequencing (ChIP-Seq) targeting the enhancer enriched transcriptional cofactor, p300

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[14]. Recently the same group has adopted a comparable epigenomic approach to identify cardiac enhancers in both the developing heart during morphogenesis and also the human

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heart under pathological conditions [15,16]. Both studies implicated the dynamic cardiovascular enhancer landscape during both cardiac morphogenesis and pathological remodelling. Using an in vitro developmental model, the epigenome has recently been

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profiled at four well defined stages during the cardiogenic differentiation of embryonic stem cells into cardiomyocytes. Through assessing the enhancer associated epigenetic marks, H3K4me1 and H3K27ac, over 80,000 putative developmental enhancers were identified [17]. Comparable to what has been observed in vivo, the sets of active enhancers were largely unique, even within closely related cardiovascular cell lineages. Furthermore, the investigators demonstrated that extremely rapid chromatin state transitions occurred at these enhancers during cardiovascular cell specification, differentiation and maturation. Using these stage specific enhancer dynamics, this study also predicted combinations of cardiac TFs that dynamically orchestrate cardiovascular cell development by examining TF motifs enriched in the stage-specific enhancer sets [17]. These and other data therefore

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ACCEPTED MANUSCRIPT demonstrate that combinatorial binding of LDTFs at enhancers provides a robust mechanism for ensuring appropriate temporal and spatial gene expression patterns during development. Disruption of enhancer activity and sequence can have wide-ranging implications for

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the correct execution of developmental and pathological gene expression programs being causative in a number of Mendelian disorders. Recent genome wide association studies

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(GWAS) have demonstrated that disruption of enhancer activity by trait associated single nucleotide variants (SNVs) is a common mechanism in complex disease [18]. A recent

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epigenomic screen in human tissues demonstrated that trait associated SNVs were enriched specifically in enhancers active in pathophysiologically relevant tissues [18]. For example,

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SNVs linked to electrocardiographic traits were enriched in enhancers predominantly active within the heart. These findings suggest that common human genetic variants linked to common disease and specific traits can contribute to physiology and pathology by affecting

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enhancer activity and the downstream gene regulatory networks hardwired by them. Stress and environmental dependant cardiac pathology is also intimately linked to the dynamic

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activity of the cardiac enhancer landscape. Two recent studies investigated the enhancer landscape during stress induced pathological remodelling within the murine heart. One study assessed the enhancer landscape, defined by H3K27ac, during pressure overload induced

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pathological remodelling [19] while the other investigated distal GATA4 bound enhancers during adult heart homeostasis [20]. Importantly, both studies demonstrate that stress dependant TF occupancy at cardiac enhancers underpins the transcriptional programs that drive the pathological response. Furthermore, the chromatin landscape at enhancers is a key determinant of the transcriptional reprogramming that underpins these pathological processes, placing enhancers as central units within these pathological gene regulatory networks. Therefore, the dynamic remodelling of the enhancer landscape appears to be a common and critical feature associated with the execution of developmental gene regulatory networks and is dysregulated within the adult heart leading to pathogenesis and disease.

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ACCEPTED MANUSCRIPT 4. Master Switches – Super-enhancers A general property associated with enhancers is the enrichment of specific proteins from the transcriptional apparatus including for example, Mediator, which are typically not

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uniformly distributed across the genome. Mediator is of particular interest and is a transcriptional coactivator protein that has been shown to be essential for enhancer

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promoter communication and interaction. Recent insights obtained from genome scale protein DNA binding data (ChIP-Seq) have demonstrated that genomic regions associated

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with extremely high density of Mediator or other transcriptional proteins have been defined as the so called SEs [18,21]. SEs were initially identified in mouse embryonic stem cells in

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the following way (1) Enhancers were considered as sites bound by all three of the ‘Yamanaka factors’, Oct4, Sox2 and Nanog, (2) Individual enhancers within 12.5Kb of each other were stitched together to define a single genomic region as an enhancer entity, (3)

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Stitched enhancers and the remaining individual enhancers were ranked based on the background normalised Mediator (Med1) enrichment within each genomic region

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(Figure.1)[21]. Once ranked it was shown that less than 3% of these enhancers contained Med1 above a point where the slope of the plot was 1 (Figure.1). These genomic regions were then defined as SEs while the remaining enhancer regions were considered typical

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enhancers (TEs). Importantly, SEs are associated with specific and unique properties when compared to TEs across the genome [18,21]. When using the current definition, SEs tend to span large genomic regions as compared to TEs, with their median size typically an order of magnitude larger than that of TEs. Furthermore, in addition to Med1, a plethora of factors typically associated with enhancer activity including increased chromatin accessibility, the histone modifications H3K27ac, H3K4me1, H3K4me2, chromatin factors such as cohesin, p300 and CBP, RNA polymerase II (RNAP2) and increased transcription of enhancer associated ncRNAs (eRNAs) show enrichment at SEs relative to TEs [22,23](Table.1). Due to these observations, SEs can also be identified on the basis of many of these unique features. Since their initial description, SE characterisation has gone beyond embryonic stem cells. A

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ACCEPTED MANUSCRIPT number of stress and lineage determining TFs have been used to identify cell specific SEs including C/EBPα in macrophages, T-bet in Th-cells, PU.1 in pro-B cells and MyoD in myotubes [24-29](Table.1). Importantly, the SEs identified in all the cell lineages spanned

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interesting genomic domains that were highly cell type specific and associated to master cell type specific regulatory genes when compared to TE associated genes and genomic regions

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[18,21,24-29]. Consistent with these observations, SEs were also found to be enriched for sequence motifs linked to master lineage determining transcription factors when compared

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to TEs, further supporting their putative important regulatory potential. Although the current evidence supports the notion that SEs represent key regulatory

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sequences for the regulation of cell identity genes, caution is required when considering absolute differences of these sequences versus TEs. These concerns are in particular related to the ways in which SEs are identified and defined. Indeed, the identification of SEs

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requires the clustering of TEs in to broader stitched enhancer domains followed by the separation of these stitched clusters into TE or SEs based on ranked enhancer associated

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bound TF or chromatin mark enrichment (Figure.1D). It is therefore challenging to interpret and separate any regulatory effects that may represent bona fide SE characteristics from those that are reflective of the embedded and clustered TEs. However, enhancer clustering

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is not sufficient or necessary for SEs which the majority of them active in pluripotent cells containing three or less stitched TEs. Furthermore, approximately 15% of SEs are composed of a single enhancer suggesting they encode unique regulatory roles independently of stitched clusters of TEs. These data therefore support the notion that SEs are not necessarily functional units composed solely of several stitched TEs. To conclude, although many fundamental questions remain to be elucidated, the concept of the SE as a novel regulatory paradigm remains and warrants further investigation and validation in future studies. Recently other transcriptional cofactors have been found to be exquisitely associated with SEs, including BRD4, a bromo-domain contacting protein that recognises acetylated lysine residues. BRD4 was found to be enriched at SEs in multiple melanoma and large B-

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ACCEPTED MANUSCRIPT cell lymphoma cells with treatment of these cells with JQ1, the BET bromo-domain inhibitor, leading to preferential loss of BRD4 at SEs. This specific preferential loss was associated with transcriptional elongation defects that preferentially impacted oncogenic genes

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regulated by SEs. It was suggested that SE associated genes confer the increased sensitivity to loss of BRD4 and thereby causes selective inhibition of transcription at

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oncogenes including Myc [30]. SEs are also critical for determining cell lineage identity and plasticity of adult stem cells in vivo. Using H3K27ac, SEs were identified in hair follicle stem

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cells and their differentiated progeny in vivo and were shown to be either repressed or activated in a lineage dependant manner. Interestingly, SE associated genes critical for

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determining stem cell fate were actively repressed by gaining the H3K27me3 mark in a process described as ‘super-silencing’. To validate the functionality of these elements, the ability of SEs to drive reporter gene expression in vivo was tested. Importantly, examination

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of the enhanced-GFP patterns in their reporter system provided compelling evidence for their functional importance in hair follicle stem cells and their differentiated progeny in vivo

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[31].

Finally, SEs were globally assessed in murine T-cells in a non-biased fashion with the aim of identifying key regulatory nodes involved in cell specification. The authors found

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that cytokine receptors and their cognate cytokines were the dominant class of genes associated with SEs in T-cells. Consistent with earlier work, disease associated SNVs for immune disorders, including rheumatoid arthritis, were highly enriched within T-cell SEs and not TEs or SEs from other cell lineages. As observed for Brd4 inhibition, treatment of T-cells with a Janus-kinase (JAK) inhibitor disproportionally altered the expression of SE associated immune disorder genes, placing SEs at the core of the gene regulatory networks underpinning these pathologies [29]. This study clearly demonstrates the power and provides a systematic approach by which the SE landscape of a relevant cell type can be integrated with human genetics to illuminate our understanding of disease and discovers novel therapeutic drug targets.

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ACCEPTED MANUSCRIPT Prior to the initial identification of SEs, Collins and colleagues identified genomic regions with comparable characteristics and described them as ‘stretch-enhancers’ [32]. These investigators used ChIP-Seq to define regulatory elements including enhancers,

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promoters and actively transcribed regions in human pancreatic islets. Importantly, this study demonstrated that previously characterised locus control regions (LCRs) are typically

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associated with ‘stretch-enhancers’ in the appropriate cell types that were linked to robust changes in transcriptional output. However, in a recent perspective it has been noted that

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there appear to be significant differences, including the absolute number, of SEs versus ‘stretch-enhancers’, suggesting major differences between the two enhancer types [22].

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However this is not surprising when considering methodological differences many of which are set in an arbitrary fashion. Ultimately, SEs and ‘stretch-enhancers’ may encompass comparable features and this requires further interrogation in the future. Finally, one of the

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most interesting characteristics associated with SEs was the enrichment of disease associated SNVs. For example, SNVs associated with type-2 diabetes were highly enriched

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in pancreas specific SEs. This was observed for a number of different tissue specific SEs and diseases including electrocardiographic variants being enriched in heart specific SEs

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and Alzheimer disease associated variants in brain specific SEs [18].

5. Cardiovascular Super-enhancers Currently there is a limited amount of data relating to cardiovascular specific SEs. However, a recent study from the Rick Young laboratory used H3K27ac ChIP-Seq data to identify SEs in 86 human cell and tissue types including the left ventricle from the adult human heart [18]. Importantly approximately 400 SEs were identified in the human adult left ventricle which were extremely heart specific when compared to left ventricular TEs. Furthermore, the associated genes to the cardiac SEs were also significantly heart enriched. Using gene ontology analysis it was shown that the cardiac SE associated coding genes were linked to biological processes that define the function and identity of the adult human heart including muscle contraction, heart development and regulation of heart contraction.

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ACCEPTED MANUSCRIPT Furthermore, to confirm the cardiac specificity of identified SEs, the authors reasoned that candidate cardiac master LDTFs should be associated with left ventricular SEs. Within the left ventricular SEs, key cardiac TFs were found to be associated with them including

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TBX20, TBX5, MEF2A, NKX2.5 and GATA4 placing SEs as key sentinel regulatory hubs within the cardiac gene regulatory network. Finally, the authors searched left ventricular SEs

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for SNVs linked to cardiovascular disease. They compiled a list a list of 5,303 SNVs linked to diverse phenotypic traits and disease in 1,675 GWAS and investigated their enrichment in

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tissue specific SEs and TEs identified within their catalogue. Interestingly, within the heart, they found that 15 non coding SNVs linked to electrocardiographic traits and atrial fibrillation

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were enriched in left ventricular SEs. Overall, these data suggest that cardiac SEs are important for regulating cardiovascular cell identity and morphogenesis via their association with key cardiovascular cell identify genes, transcription factors and common SNVs linked to

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cardiovascular traits and disease [18].

Recent studies in cancer cell types have demonstrated that JQ1, a BET inhibitor able

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to inactivate Brd4, led to reduced occupancy of Brd4 at SEs and to decreased expression of SE associated oncogenes including Myc [30]. In a mouse model of multiple myeloma JQ1 inhibition of Brd4, and the downstream target gene Myc, had potent therapeutic anti-

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proliferative effects. Therefore, the connection between oncogenic SEs and Brd4 can lead to a specific therapeutic response, which in this case was to inhibit multiple myeloma associated uncontrolled cellular proliferation. Interestingly, Brd4 and the BET domain inhibitor JQ1, have recently been implicated in the global transcriptional reprogramming that underpins pathological cardiac remodelling and heart failure [33]. The investigators demonstrated that Brd4 inhibition via JQ1, potently suppressed cardiomyocyte hypertrophy in vitro and pathological remodelling leading to heart failure in vivo. Furthermore, they utilised genome-wide approaches to demonstrate that Brd4 functions mechanistically as a pause-release factor for the expression of key cell identity genes that are central to heart failure pathogenesis and linked to the pathophysiology in the failing human heart. These data clearly demonstrate that BET bromodomain reader proteins like Brd4, which is enriched

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ACCEPTED MANUSCRIPT at SEs, are indispensable coactivators in the transcriptional reprogramming that underpins pathological remodelling and heart failure. More importantly, considering the previous observation that assymetrically loaded Brd4 is enriched at cell specific SEs, this data raise

important regulatory functions for cardiovascular biology [33].

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the possibility that heart specific SEs may be active in the adult stressed heart and encode

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Despite the fairly limited understanding of the identity and roles of cardiac SEs during cardiovascular development and disease, a number of human and mouse ChIP-Seq

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datasets exist that may allow the illumination of the cardiovascular SE landscape. These include datasets from the developing human and mouse heart [14-16], differentiating

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embryonic stem cells [2] and adult human and mouse hearts undergoing pathological stress, remodelling and failure [19,20]. These datasets utilised various TFs and chromatin states to assess enhancer dynamics, all of which are amenable to SE characterisation. Future

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retrospective computational analysis of these datasets promises to expand our understanding of the SE landscape within cardiovascular cells and tissues in various

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developmental and pathological contexts.

6. Super-enhancer associated lncRNAs

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One of the unique observations specific to SEs was their increased transcription and production of enhancer associated ncRNAs (eRNAs, elncRNAs). Enhancer associated lncRNAs are typically transcribed from genomic regions harbouring canonical enhancer specific chromatin states, H3K4me1 and H3K27ac and associated cofactors (i.e.p300). Importantly, pioneering studies demonstrated that elncRNA expression is coupled with developmental and signal dependant transcriptional changes of their target genes [34-36]. These transcripts primarily exist as two primary transcripts. The first type described, the eRNAs, are bidirectionally transcribed, non-polyadenylated and extremely common, found at most active enhancers [37]. The other, albeit less common, is unidirectional, multiexonic, spliced and polyadenylated, resembling the previously identified intergenic lncRNAs (lincRNAs), and typically known as enhancer associated lncRNAs (elncRNA)[38]. Upon their

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ACCEPTED MANUSCRIPT initial discovery, controversy persisted as to whether enhancer associated lncRNAs were required for enhancer function. However, a plethora of recent functional studies utilising sophisticated –loss (LoF) and –gain (GoF) of function approaches have demonstrated that

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elncRNAs are required for enhancer activity and target promoter transcriptional activity [28,38-43].

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Initial LoF studies demonstrated that elncRNAs are required for enhancer promoter communication and chromatin looping contributing to the initiation and stabilisation of the

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loops. This allows the enhancers to be integrated within the gene regulatory networks hardwired by them [28,39]. However, early studies also suggested that enhancer associated

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transcripts were not required for this looping process, raising doubts to their mechanism of action. An alternative mechanism of action was recently proposed demonstrating that elncRNAs may in fact function once the chromatin loop is already established via facilitating

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RNAP2 pause-release at target transcriptional starts, thereby promoting transcriptional elongation [43]. Furthermore, another recent study demonstrates that SE associated

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lncRNAs interact with divergently transcribed RNA exosome-senstive lncRNAs at both promoters and enhancers to facilitate the formation of integrated chromatin loops and nuclear topological domains promoting the expression of target genes [44]. All mechanisms

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of action determined to date primarily suggest that elncRNAs function in a -cis dependant manner which is consistent with their low expression levels, their absence at genomic regions other than their site of transcription and finally the minimal effects of LoF on nonadjacent protein coding genes. However, this does not preclude potential -trans acting regulatory functions which is supported by the observation that depletion of specific elncRNAs leads to global gene expression changes greater than that expected for the target genes function alone. In light of the ability for elncRNAs to reorganise the nuclear genomic architecture, -trans regulatory functions may be as a consequence of the ability of elncRNAs to stabilise the genome in toplogical 3D domains therefore favouring the acquisition of particular cell fates during development. The integrated remodelling of such topological

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ACCEPTED MANUSCRIPT domains is likely to be of importance for the hardwiring of appropriate down-stream cardiovascular gene regulatory networks. Not surprisingly, a number of SE associated lncRNAs have recently been functionally

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and mechanistically characterised. For example, during myogenesis there is an extremely large SE domain encompassing the MyoD master myogenic TF. In a recent study it was CE

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shown that this SE generates two lncRNAs,

expression of MyoD and Myogenin respectively [28]. Both of these SE associated lncRNAs

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potentiated RNAP2 occupancy at their target promoters. Although the mechanism was not described, the authors suggested the SE associated lncRNAs achieved this at the level of

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chromatin remodelling by either (1) targeting chromatin to facilitate nucleosome reorganistion or (2) recruiting ubiquitously expressed chromatin modifiers and remodellers (i.e. SWI/SNF complex). The authors concluded that myogenic SEs, and perhaps others,

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served as templates for numerous lncRNAs that in concert with master regulators define cell type specific gene regulatory networks. In another study, a human colorectal cancer specific

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SE associated lncRNA, CCAT-L, was shown to regulate long range chromatin interactions at the MYC locus [45]. Knockdown of CCAT-L reduced chromatin looping between its associated SE and the MYC locus and this interaction was dependant on binding with the

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looping associated chromatin factor, CTCF. Importantly, through using genome editing technologies (TALENS), these authors also demonstrated that in -cis over-expression of CCAT-L at its native genomic locus enhanced MYC expression thereby promoting tumourogenesis. Finally, it has recently been shown that a distal TE associated lncRNA (lncRNA-CSR) interacted with SE associated lncRNAs to mediate long range DNA interactions at the IgH locus. CRISPR-Cas9 mediated depletion of this lncRNA decreased chromatin looping within the locus leading to decreased class switch recombination efficiency. The authors conclude that SE and TE associated lncRNAs interact to mediate important SE chromosomal interactions important for cellular fates and functions [44]. Despite these encouraging early studies, the mechanisms governing SE associated lncRNA expression are poorly understood. An important insight has recently been made by

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ACCEPTED MANUSCRIPT the Shiekhattar group implicating the RNAP2 associated Integrator protein with the biogenesis of SE associated lncRNAs [46]. They demonstrate that Integrator is recruited to SEs in a stimulus dependant manner with depletion associated with reduced enhancer

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promoter chromatin looping and communication. Furthermore, SE and target gene expression requires the catalytic activity of Integrator conferring a critical role for this protein

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7. Cardiovascular SE-associated lncRNAs

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in SE associated lncRNA biogenesis and function in metazoans.

Considering the identification of cardiovascular SEs is still in its infancy, it is of no

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surprise that well characterised examples of associated lncRNAs in the cardiovascular system are lacking. There are however some emerging examples of lncRNAs within the heart, many of which are derived from SE loci that may have functional and regulatory roles

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which we will briefly discuss here. Our group set out to investigate this landscape within the remodelling murine heart post myocardial infarction (MI). We utilised a very-deep RNA-

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sequencing (RNA-Seq) approach followed by de novo transcript reconstruction to profile the mouse long noncoding transcriptome post MI [47,48]. We identified approximately 1500 completely novel previously unannotated lncRNAs, many of which were differentially

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expressed in the stressed heart. To identify what portion of these were derived from the enhancer landscape, we integrated publicly available ChIP-Seq data sets and demonstrated that the vast majority were derived from active cardiac enhancers in the adult mouse heart. Furthermore, those that were modulated post-MI were significantly more enriched with enhancer sequences implicating the enhancer landscape and associated lncRNAs in the transcriptional reprogramming that underpins the pathological response. Using chromatin state based enhancer dynamics during cardiac differentiation [17], many functions were inferred for these lncRNAs. Interestingly, most of the inferred functions were linked to fundamental cardiac processes including those linked to developmental, structural and functional cardiovascular gene programs. Interestingly, this observation is comparable to that observed for genes associated with SEs in various cell and tissue types.

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ACCEPTED MANUSCRIPT Considering that the reactivation of the fetal gene program is a hallmark of the remodelling heart, it is likely that enhancer associated lncRNAs that are modulated post-MI activate specific biological processes as an attempt to re-active a developmental program.

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One such novel enhancer associated lncRNA, Novlnc6, was shown to be derived from a bona fide fetal cardiac developmental enhancer and linked via enhancer chromatin dynamics

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to key developmental cardiac gene programs [47]. Depletion of this elncRNA directly affected two predicted critical cardiac target genes, Bmp10 and Nkx2.5 supporting the

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importance of identified enhancer associated lncRNAs. To translate these findings to human, we mapped the novel mouse lncRNAs to the human genome identifying hundreds of human

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orthologs using TransMap. TransMap is a cross species mRNA alignment tool using syntenic BLASTZ alignments that consider conserved gene order and synteny. Importantly, those that were validated, including NOVLNC6, were differentially expressed in diverse

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cardiac pathologies including dilated cardiomyopathy and aortic stenosis. These results demonstrate that many of the enhancer associated lncRNAs and down stream gene

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regulatory networks were also conserved in human. Considering that many of our novel enhancer associated lncRNAs were proximal to important cardiovascular cell identity genes, were were curious to determine if these were mapping to previously identified cardiovascular

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SEs. We therefore mapped our novel Transmapped human lncRNAs to the left ventricular TE and SE catalogs generated by the Rick Young laboratory [18] and interestingly found that 32% of human left ventricular SEs were associated with novel lncRNAs compared to only 5% of the left ventricular TEs (Ounzain et al, unpublished). This suggests that many of our novel lncRNAs are bona fide SE associated lncRNAs and further suggests that SEs are more likely to encode multiexonic, polyadenylated and unidirectional lncRNAs as compared to TEs. We suspect this characteristic may have functional implications for the observed master cell identity regulatory roles attributed to SEs. Potentially, -trans acting lncRNAs derived from SEs, could expand the functional repertoire for SEs increasing their genomewide regulatory potential. Interestingly, many critical important master TFs and regulatory proteins within the heart, including TBX20 and MEF2A were associated with SE associated

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ACCEPTED MANUSCRIPT novel lncRNAs [47](Ounzain et al, unpublished). These transcripts represent interesting candidates for further functional and mechanistic characterisation within the context of adult heart homeostasis and pathological remodelling.

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SEs were initially described in stem cells and their derived developmental lineages. It is therefore of importance to identify developmental cardiac SE associated lncRNAs. Our

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laboratory has recently characterised transcription from bona fide cardiac developmental enhancers previously identified in epigenomic and transgenic reporter screens [14,36]. The

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elncRNAs we identified were dynamically expressed in a correlative manner with their predicted target genes consistent with a -cis regulatory mode of action. Furthermore,

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depletion of candidate elncRNAs was associated with down regulation of their target genes. Many of these newly identified elncRNAs were derived from enhancers regulating key cell identify genes, a property associated with SEs. For example, an elncRNA derived from the

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enhancer mm85 controls the expression of the key cell identity gene Myocardin. This is a transcriptional cofactor that interacts with serum response factor (SRF), a master TF integral

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to the cardiac gene regulatory network [49]. To globally asses the long noncoding transcriptome associated with developmental enhancers, very-deep RNA-Seq was executed on PolyA(+) RNA derived from mouse embryonic stem cells differentiating into

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cardiomyocytes. Computational reconstruction of the transcriptome identified hundreds of novel lncRNAs derived from developmental cardiac enhancers that undergo lineage and state specific transitions during cardiac specification and differentiation. Manual interrogation of these novel lncRNAs identifies many linked to putative SEs proximal to important developmental cell identity genes which warrants further investigation (Ounzain et al, unpublished)[36]. One of the first identified and characterised lncRNAs in cardiovascular development was Braveheart (Bhvt), a lncRNA transcribed from a very important miRNA containing developmental locus [50]. Despite not being directly associated with an enhancer sequence, this locus encodes a very powerful left ventricular specific SE in the adult human heart [18] and also a SE enhancer like signature in differentiated cardiovascular progenitors in mouse

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ACCEPTED MANUSCRIPT embryonic stem cells [17]. Bhvt expression is induced during cardiogenic differentiation of ES cells but is also expressed at high levels in adult heart tissue suggesting that it represents an important lncRNA for cardiovascular lineage specification, differentiation and

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maturation. Confirming this hypothesis, Bhvt LoF perturbed the ability of embryonic stem cells to differentiate into cardiomyocytes. Within the cardiac gene regulatory network, Bhvt

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appeared to function upstream of the key cardiac mesoderm specifying TF, Mesp1, an important TF that marks early cardiac mesoderm and activates the cardiovascular gene

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regulatory network during development. The authors propose that through this modulation of the cardiac gene regulatory network, Bhvt dictates the lineage transition from nascent to

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cardiac mesoderm, which then generates differentiated cardiomyocytes. This seminal finding, for the first time, demonstrated that lncRNAs could represent powerful regulatory molecules capable of inducing cardiac specification and differentiation. However, a Bvht

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ortholog was not found in human which suggested other cardiac specifying lncRNAs likely exist.

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Our group was particularly interested in the human SE-containing orthologus locus and identified another, SE associated lncRNA that was conserved in human and named (CAR)diac (M)esoderm (E)nhancer associated (N)oncoding RNA, CARMEN [51]. CARMEN

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was identified in a high-throughput screen to identify the most significantly up-regulated lncRNAs during the cardiac differentiation of human cardiac precursor cells isolated from the fetal human heart. CARMEN was of particular interest because it was associated to an active SE in both the fetal and adult human heart, in addition to its proximity to the key cardiovascular miRNA-143 and -145 containing cell identity locus [52]. We demonstrated that CARMEN is a bona fide SE associated cardiovascular lncRNA that exhibits RNA dependant enhancing activity and is upstream of both Bvht (in mouse) and the cardiac mesoderm specifying gene regulatory network. CARMEN LoF abolished the ability of precursor cells to differentiate into cardiomyocyes suggesting that CARMEN is an evolutionary conserved regulator of cardiovascular cell lineage specification and differentiation. Finally, a number of other important lncRNAs have also recently been

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ACCEPTED MANUSCRIPT identified and characterised that have been show to play important roles in cardiovascular cell specification and differentiation including Chrf [53], SENCR [54], Fendrr [55,56], PUNISHER, ALIEN and TERMINATOR [57](Figure.2).

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Many cardiac SEs are not intergenic, and typically overlap coding and intronic sequences of key cell identity genes. Interestingly, an important SE containing cardiac cell

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identity and structural gene, myosin heavy chain 7 (Myh7), has recently been shown to encode an overlapping SE associated lncRNA named Myheart (Mhrt)[58]. This lncRNA is

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extremely abundant in adult human and mouse hearts and has been implicated in maintaining cardiac function within the stressed heart. Pathologically inducing stressors

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inhibit Mhrt expression with this repression critical for the development of cardiomyopathy. Importantly, the authors demonstrate that forced expression (GoF) of Mhrt, using a transgenic approach, is sufficient to protect the heart from remodelling associated

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cardiomyocyte hypertrophy and subsequent heart failure. At the molecular level, Mhrt functions by antagonising the activity of Brg1, a chromatin remodelling factor that had

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previously been implicated in the control of pathological cardiovascular gene regulatory networks. Mhrt directly interacts as a molecular decoy, titrating Brg1 away from its genomic binding sites therefore suppressing the pathological gene program. Importantly, human

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MHRT is also significantly depleted in the hearts of patients suffering from cardiomyopathy, supporting a conserved regulatory role in human cardiovascular disease.

8. Conclusion With the advent of new high throughput genomic and epigenomic technologies the community is forging new frontiers in the understanding of the enhancer landscape and associated lncRNAs in cardiac development and disease. In particular SEs and their associated transcripts are emerging as powerful -cis regulatory sentinel hubs with important roles hardwiring the gene regulatory networks underpinning cardiovascular cell identity, development and ultimately disease. SE associated lncRNAs appear to contribute to the activity and specificity of the SEs that produce them. This therefore raises the therapeutic

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ACCEPTED MANUSCRIPT possibility to modulate SE activity by targeting the lncRNAs produced by them, therefore providing a means to control context specific expression of protein coding genes in a highly cell and tissue type specific manner in vivo. For example in patients suffering from heart

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failure, one could therapeutically target the pathological SEs to perturb the activation of pathological gene regulatory networks and improve clinical outcomes. One could envisage

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using nuclear-active modified anti-sense oligoncleotides (i.e. GapmeRs) to deplete pathological SE associated lncRNAs. For instance, cardiac fibrosis could be inhibited

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targeting SE associated lncRNAs linked to cardiac fibroblast specific gene expression and activation. Alternatively, cardiac hypertrophic SEs, akin to the Mhrt encompassing locus,

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could be therapeutically modulated to perturb cardiac hypertrophy. Ultimately, through manipulating key cell identify associated SEs via their transcribed lncRNAs, it may also be possible to switch the pathological reparative response to a more regenerative process, the

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‘holy grail’ of cardiac regenerative medicine. Cardiac regeneration in zebrafish and neonatal mammals is associated with the de-differentiation of mature CMs, disassembly of the

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sarcomere and subsequent re-entry into the cell cycle allowing the proliferation of preexisting CMs. One could therefore envisage targeting SE associated lncRNAs linked to maintaining CMs in a mature non-dividing and those that repress the entry of the CM into the

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cell cycle. Depletion of these transcripts could reprogram the gene regulatory networks enforcing this states potentially allowing CMs to de-differentiate and proliferative, promoting a cardiac regenerative response. It is of importance that the cardiovascular research community further increases our understanding of the language and function of both cardiovascular SEs and their associated lncRNAs. This greater understanding will provide valuable insights into the molecular mechanisms governing cardiovascular cell development and pathological remodelling, hopefully identifying novel first in class disruptive therapeutic targets. The SE landscape with its associated noncoding transcriptome has unprecedented potential for drug discovery and will remain a rich field of investigation for many years to come.

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ACCEPTED MANUSCRIPT Sources of funding This work is in part funded by a grant from the Swiss National Science Foundation within the frame of the National Research Program 63 on “Stem cells and Regenerative Medicine” (TP;

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Grant no 406340-128129).

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Author contribution

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SO wrote the paper; TP created the figures and edited the manuscript.

Disclosure statement

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A patent application covering the therapeutic use of lncRNAs has been filed, and is pending. PCT application PCT/EP2014/078868 filed on December 19, 2014, based on US provisional

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Ounzain and T. Pedrazzini.

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Patent application No. US 61/964,591 filed on December 20, 2013, naming as inventors S.

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Figure 1. Identification and characteristics associated with super enhancers. (A & B) SEs are composed of stitched active typical enhancers which are marked at the chromatin level with high H3K27Ac, H3K4me1 and low H3K4me3. (C) Stitched enhancers

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are ranked based on enrichment of appropriate factor and those above a point on slope that is >1 are considered SEs. (D) SEs can potentially be identified in individual cardiovascular cell types including cardiomyocytes and cardiac fibroblasts.

Figure 2. Developmental cardiovascular lncRNAs. Illustration of currently functionally characterized cardiovascular lncRNAs and the associated developmental pathways and differentiated cell types they are associated with.

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H3K27Ac

[18,62]

TBET

[63]

GATA3

[63]

ROR

[63]

STATs

[63] [64]

NFKB

[24,65]

KLF

[64]

SIM2

[66]

TEX10

[67]

P300

[29]

MEDIATOR

[21]

PU.1

[27]

CEBPs

[27] [64]

PBX1

[64]

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ATF

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AP1

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[59-61]

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BRD4

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Reference

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Factor

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Table.1 Factors and chromatin marks used to identify Super Enhancers

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Highlights

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 Cardiac development and response to stress depend on the dynamic control of tissue-specific gene expression by distant acting transcriptional enhancers

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 Super-enhancers are emerging as sentinel regulators within the cardiac gene regulatory networks

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 Long noncoding RNAs associated with enhancers play fundamental roles for their activity

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 Exploration of this genomic landscape provides novel therapeutic targets for the amelioration of cardiovascular disease

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